Low genetic variation and high
differentiation across sky island
populations of Lupinus alopecuroides
(Fabaceae) in the northern Andes
Diana L. A. Vásquez, Henrik Balslev,
Michael Møller Hansen, Petr Sklenář &
Katya Romoleroux
Alpine Botany
ISSN 1664-2201
Alp Botany
DOI 10.1007/s00035-016-0165-7
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DOI 10.1007/s00035-016-0165-7
ORIGINAL ARTICLE
Low genetic variation and high differentiation across sky island
populations of Lupinus alopecuroides (Fabaceae) in the northern
Andes
Diana L. A. Vásquez1 • Henrik Balslev2 • Michael Møller Hansen2
Petr Sklenář1 • Katya Romoleroux3
•
Received: 18 November 2015 / Accepted: 11 April 2016
Ó Swiss Botanical Society 2016
Abstract The tropical alpine flora in the northern Andes
has caught the attention of evolutionary biologists and
conservationists because of the extent of its diversity and
its vulnerability. Although population genetics studies are
essential to understand how diversity arises and how it can
be maintained, plant populations occurring above 4100 m
a.s.l. in the so-called super-páramo have rarely been studied at the molecular level. Here, we use 11 microsatellite
DNA markers to examine genetic structure in populations
of Lupinus alopecuroides, a long-lived semelparous giant
rosette known from only 10 geographically isolated populations. Each population is located on a different
mountain top, of which three are in Colombia and seven in
Ecuador. We analysed 220 individuals from all the ten
known populations. We find low genetic variation in all but
one of the populations. Four populations are completely
monomorphic, and another five show only one polymorphic locus each. On the other hand, we find extremely high
genetic differentiation between populations. We discuss the
mechanisms that might cause this pattern, and we suggest
that it is related to founder effects, lack of gene flow, and
Electronic supplementary material The online version of this
article (doi:10.1007/s00035-016-0165-7) contains supplementary
material, which is available to authorized users.
& Diana L. A. Vásquez
[email protected]
1
Department of Botany, Charles University, Benátská 2,
128 01 Prague 2, Czech Republic
2
Department of Bioscience, Aarhus University, Ny
Munkegade 116, Building 1540, 8000 Aarhus C, Denmark
3
Escuela de Ciencias Biológicas, Pontificia Universidad
Católica del Ecuador, Av. 12 de Octubre y Roca Apdo. 2184,
Quito, Ecuador
autogamy. The genetic relationships among the populations, and the lack of correlation between the genetic and
geographic distances also point to the importance of
founder effects and colonization history in driving differentiation among the populations.
Keywords Population genetics Genetic diversity Founder effects Páramo Andean flora Lupinus alopecuroides
Introduction
The highlands in the northern Andes host one of the World’s
most species-rich tropical alpine floras (Smith and Cleef
1988; Sklenář et al. 2011), characterized by rapid and recent
evolution (van der Hammen et al. 1973; Hughes and Eastwood 2006; Madriñán et al. 2013). This is the so-called
páramo ecosystem that lies between 3000 and 4900 m a.s.l.,
and the highest tiers of it, above 4100 m, is called the superpáramo (Cuatrecasas 1968; van der Hammen and Cleef
1986). Given its restricted geographic distribution, páramo
plant diversity is severely threatened by habitat destruction
due to global warming and land-use changes (Balslev and
Luteyn 1992; Morueta-Holme et al. 2015; Vásquez et al.
2015). Although the páramo flora has received significant
attention from evolutionary biologists and conservationists,
population genetic studies are scarce (Oleas et al. 2012). To
our knowledge, super-páramo plant populations have never
been studied at the molecular level.
Patterns of genetic variation across populations are
determined by various interrelated factors, and population
history plays an important role (Ellstrand and Elam 1993;
Hewitt 2000). In the northern Andes, Pleistocene climatic
oscillations produced elevational range shifts that
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enormously impacted páramo species. During glaciations,
species expanded across broader landscapes at lower elevations, establishing new populations and gene flow
between populations that had been isolated. During interglacials, species were again restricted to geographically
isolated ‘‘islands in the sky’’ (van der Hammen et al. 1973;
Hooghiemstra et al. 2006; Graham 2009; Sklenář et al.
2011). Another historical factor that impacted the páramo
populations is volcanism (Oleas et al. 2012). Volcanic
activity is a driver of population extinction and recolonization (Carson et al. 1990; Beheregaray et al. 2003), and thus
influences evolutionary trajectories of the populations.
We examined the genetic structure of populations of
Lupinus alopecuroides Desr.—a long-lived semelparous
giant rosette restricted to the super-páramo in Colombia and
Ecuador. Semelparous giant rosettes are a distinctive feature
of tropical alpine floras, and a fascinating example of convergent adaptive evolution to harsh high-elevation
environments (Rundel et al. 1994; Sgorbati et al. 2004).
Marcescent dead leaves of the rosette protect the meristem
from nocturnal temperatures, which often are below zero
(Monasterio 1986; Rundel et al. 1994), and lanate hairs on
the up-to 1 m high inflorescence provide insulation for
reproductive organs (Miller 1986) (Fig. 1). Semelparous
plants maximize reproduction by investing all resources in a
single reproductive episode because, due to harsh environmental conditions, future survival and reproduction are less
likely (Young and Augspurger 1991).
The genetic structure of populations is also determined by
species-specific biological traits such as dispersal ability and
breeding system. One of the most common seed dispersal
modes observed in lupines is ballistichory—a mechanical
process in which the walls of the mature pod twist in
opposite directions, firing the seeds 1–3 m away from the
parent plant. Long-distance dispersal of lupine seeds is
usually afforded by waterways, human activities, and animals, mainly rodents (Milla and Iriondo 2011; Fagan et al.
2005; Maron and Kauffman 2006; Australian Department of
Health and Ageing 2013). The reproductive biology of L.
alopecuroides has remained unstudied. Other perennial
species of Lupinus have asexual reproduction by vegetative
regeneration, and there is no evidence of apomictic reproduction in the genus (Richards 1986). Most annual lupines
are self-compatible while perennials are self-incompatible
(Kittelson and Maron 2000). However, in the papilionoid
legumes, to which Lupinus belongs, self-sterility is rare
(East 1940; Kittelson and Maron 2000). For facultatively
autogamous species, selfing may occur either as a result of
insufficient pollinator visitation, or as a result of pollinator
visits due to the close proximity of viable pollen to the
receptive stigma (Pazy 1984; Karoly 1992), or due to
transfer of pollen between flowers on the same individual
(de Jong et al. 1993).
Another important factor influencing the genetic structure
of natural populations is landscape (Slatkin 1987; Manel and
Holderegger 2013). Populations of Lupinus alopecuroides
are known from only ten geographically isolated mountain
tops (Fig. 2). In geographically isolated populations,
enhanced genetic drift and lack of gene flow result in genetic
differentiation and loss of genetic diversity (Lesica and
Allendorf 1995; Frankham et al. 2002; Allendorf and
Fig. 1 Typical habit and local distribution of Lupinus alopecuroides
in the superpáramo of Sincholagua, Ecuador (Photo by Diana L.
A. Vásquez)
Fig. 2 Geographic distribution of 10 locations (mountain tops),
where populations of Lupinus alopecuroides were sampled. Map by
Flemming Nørgaard
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Luikart 2006). In these tropical ‘‘islands in the sky’’, founder
effects may also occur, as founders arrive over long distances from similar habitats, rather than from adjacent sites
at lower elevations, which are ecologically different (Carson
and Templeton 1984). Enhanced genetic drift, restricted
gene flow, and founder events put the sky island populations
of L. alopecuroides at risk of inbreeding depression, and
potentially make them vulnerable to an extinction vortex
(Frankham et al. 2002). In populations of Puya raimondii,
which is another sky island long-lived semelparous giant
rosette occurring around 4000 m a.s.l from Peru to northern
Bolivia, low genetic variation was reported across 217
AFLP marker loci in 160 individuals from eight populations.
Four populations were completely monomorphic, and each
of the others displayed only one to three polymorphic loci. It
was suggested that the causes of this pattern are high rates of
selfing and repeated bottlenecks (Sgorbati et al. 2004).
Phaedranassa tunguraguae, which is a long-lived, partially
selfing plant, showed homozygote excess at 12 microsatellite loci. Its populations were restricted to a single valley in
the Ecuadorian Andes, at 1500–2500 m a.s.l., and the most
likely explanation suggested for this pattern was genetic
drift due to small population size and restricted gene flow
(Oleas et al. 2012).
It is well established that founding of a new population by
a small number of individuals causes abrupt changes in
allele frequencies that can lead to loss of genetic variation
within populations and increased genetic differentiation
among populations (Mayr 1954, Allendorf and Luikart
2006). However, founder effects are hard to detect, because
it is difficult to disentangle their signatures from those of
other evolutionary forces (Hoeck et al. 2010; Spurgin et al.
2014). In island populations of Berthelot’s pipit (Anthus
berthelotii), Spurgin et al. (2014) detected genetic and
phenotypic changes that resulted from founder events, and
suggested that, under lack of gene flow and selection,
founder effects can persist for evolutionary time scales. In
populations of silvereyes (Zosterops lateralis), Clegg et al.
(2002) demonstrated that sequential founder events produced divergence among populations. Finally, Prugnolle
et al. (2005) have suggested that patterns of neutral genetic
differentiation in human populations are the result of
sequential founder events that occurred as small populations
of modern humans migrated out of Africa.
Population genetics studies can help us understand how
contemporary diversity arises and how it can be maintained
(Frankham et al. 2002; Allendorf and Luikart 2006). Here,
we use microsatellite markers to examine patterns of genetic
variation in the sky island populations of Lupinus alopecuroides, and we discuss the factors underlying genetic
variation and population structure. In particular, we address
the following questions. (1) How high is genetic diversity within populations? (2) Are populations genetically
differentiated, and how is genetic variation distributed
within and among the populations? (3) Is there gene flow
between the populations? (4) How are the different populations related, and is there a geographical correlation?
Materials and methods
The species
Lupinus alopecuroides Desr. is a long-lived semelparous
giant rosette known from only ten geographically isolated
populations. Each population is located on a different
mountain top, of which three are in Colombia and seven in
Ecuador (Fig. 2). Within the populations, individuals are
found forming dense clusters (Fig. 1). This might be related
to the species’ ballistic seed dispersal. We assume that longdistance dispersal of L. alopecuroides seeds, which weigh
approx. 40 mg and measure 5 9 3 mm, is limited. Elsewhere rodents and human activity are important dispersers
of Lupinus (Milla and Iriondo 2011; Fagan et al. 2005,
Maron and Kauffman 2006; Australian Department of
Health and Ageing 2013), but these agents are nearly absent
in the super-páramo (Sklenář and Ramsay 2001, Rangel
2006). Anemochory or epizoochory are also unlikely in L.
alopecuroides, since the seeds have a smooth surface without appendages or other adaptations that could facilitate
external transport by animals. Some long-distance dispersal
may be provided by waterways, white-tailed deer (Odocoileus virginianus), and domestic cattle, which have been
seen to feed on L. alopecuroides pods (Vásquez, personal
observation). The reproductive biology of the species has
remained unstudied. Bumblebees are the most likely pollinator of L. alopecuroides, but their activity may be reduced
by the harshness of the environment where this species
grows (Dillon et al. 2006). During our fieldwork for this
(10 weeks) and previous studies, we never observed insects
visiting the flower, even on the sunniest days. We also
observed that flowers remain closed for a long time after
anthesis.
Population sampling
Exhaustive field and herbarium work in 2013–2014 permitted us to sample all ten known populations of L.
alopecuroides, each of them located on a different mountain
(Fig. 2). On the Cayambe and Sincholagua mountains,
populations consist of three subpopulations clearly separated by 1–10 km (Table 1). Population size, elevation, and
slope were recorded for each population and subpopulation
(Table 1). Population size was estimated by counting all
individuals or, when population size exceeded 50, by
extrapolating it from the average number of individuals
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Table 1 Lupinus alopecuroides populations and subpopulations with
their respective population size, sample size, elevation, slope, area,
mean allelic richness per loci, observed heterozygosity (Ho), expected
Population
Subpopulation
Pop.
Size
Sample
size
Elevation
(m a.s.l.)
heterozygosity (He), and number of polymorphic loci among the 11
analysed loci
Slope
(°)
Area
(m2)
Mean allelic
richness
Ho
He
No. polymorphic
loci/pop
*50
14
4150–4300
0–10
400,000
2.72
0.144
0.51
11
RUIZ
*400
22
4450–4600
20–30
35,000
1.08
0.01
0.04
1
PURACÉ
*600
23
4100–4200
20
22,000
1.08
0
0.04
1
*60
19
4340–4350
30
700
1.11
0
0.036
1
COCUY
COTACACHI
IMBABURA
CAYAMBE
10
10
CAY1
*80
8
CAY2
*300
50
CAY3
4350–4370
35
150
1
0
0
0
4468
0–20
22,500
1
0
0
0
4425–4450
35
35,000
9
9
4100–4200
40
PICHINCHA
*250
18
4550–4600
20–30
ANTISANA
*50
12
4400–4450
SINCH1
SINCH2
*100
*40
19
9
SINCH3
*100
8
SINCHOLAGUA
CHIMBORAZO
300
38,000
1.09
0
0.04
1
35
600
1.16
0
0.06
1
4430–4438
4430–4440
30
35
5000
250
1
0
0
0
11
4490–4495
40
4500
8
4500–4600
10–20
1
0
0
0
300,000
Populations are sorted by latitude
counted in sub-plots. Population area was estimated from the
polygon obtained from GPS-delimited boundaries using
Google Earth Pro. Leaf tissue was randomly collected from
8 to 67 clearly distinct individuals, and stored in silica gel.
Molecular methods
DNA was extracted from leaf tissue of 220 individuals. Nine
loci (Luna1, Luna3, Luna4, Luna6, Luna8, Luna12, Luna15,
Luna17, Luna20) developed for Lupinus nanus (Molecular
Ecology Resources Primer Development Consortium et al.
2012), and two loci (AG55-20-22, AG55-26-16) developed
for Lupinus microcarpus (Drummond and Hamilton 2005)
were amplified in four separate multiplexes, using QIAGEN
Multiplex PCR Maxter Mix (Qiagen, Valencia, CA, USA).
PCR products were electrophoresed on an automated capillary sequencer (3130xl Genetic Analyzer, Applied
Biosystems, Foster City, CA, USA) with Genescan-600
(LIZ) size standard (Applied Biosystems). Sizes of alleles
(in base pairs) were determined using GeneMarker (Soft
Genetics).
Statistical analysis
To estimate within-population genetic diversity, allelic
richness as well as observed heterozygosity (Ho), and
unbiased expected heterozygosity (He) according to Nei
(1987) were calculated for each population. Genetic differentiation among all populations was estimated using Weir
and Cockerham’s h (1984), an estimator of FST, and RST, an
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FST analogue based on the stepwise mutation model (Slatkin
1995). The program FSTAT 2.9.3 (Goudet 2001) was used
for these analyses. Tests for significant deviation from
Hardy–Weinberg equilibrium were conducted using exact
tests implemented in Arlequin 3.5 (Excoffier and Lischer
2010). Genetic relationships among the populations were
estimated using POPTREEW (Takezaki et al. 2014) to
construct a neighbour-joining phenogram based on dmu2
distances (Goldstein et al. 1995). Bootstrap values across
loci were based on 10,000 permutations by locus. Spatial
genetic structure was further explored by testing for isolation by distance using a Mantel test between a matrix of
dmu2 genetic distances (Goldstein et al. 1995) and a matrix
of geographic distances as implemented in R package adegenet 2.0.0 (Jombart and Ahmed 2011).
Results
A total of 46 unambiguously scorable and reproducible
alleles ranging from 75 to 346 bp were detected at 11
microsatellite loci across 220 individuals, representing 10
populations distributed throughout the species’ geographic
range. Numbers of alleles per locus ranged from 2 to 6.
Monomorphic loci were observed in all the populations
except COCUY, where all eleven loci were polymorphic.
Five populations showed only one polymorphic locus each.
The other four populations (CHIMBORAZO, IMBABURA,
CAYAMBE, SINCHOLAGUA), were completely
monomorphic (Table 1). Consequently, there was no
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genetic variation between the subpopulations neither in
CAYAMBE nor in SINCHOLAGUA. Moreover,
CAYAMBE and SINCHOLAGUA were fixed for the same
alleles (Online Resource 1). Therefore, all individuals
within these populations were identical multilocus genotypes. Significant deviation from Hardy–Weinberg
equilibrium was found across all non-monomorphic loci
except Luna 3 (p value = 0.23) and Luna 17
(p value = 0.02) in the population COCUY. Genetic differentiation among all populations was high: RST = 0.65
and h = 0.84 (95 % CI 0.753–0.900).
The neighbour-joining phenogram (Fig. 3) constructed
from dmu2 distances (Goldstein et al. 1995) grouped all
Ecuadorian populations except ANTISANA in a well-supported group (65 % bootstrap value). COTACACHI,
CAYAMBE and SINCHOLAGUA were also a group with
high bootstrap value (65 %). Test of isolation by distance
yielded a non-significant outcome (p value = 0.416).
Discussion
Genetic diversity and genetic differentiation
Our analyses revealed extremely low genetic diversity in
populations of L. alopecuroides throughout its geographic
range. From the total of ten known populations, four were
completely monomorphic (all individuals within the population were identical multilocus genotypes) and another five
showed only one polymorphic locus each (Online Resource
1). On the other hand, the high h and RST values show that the
ten populations of L. alopecuroides are highly genetically
differentiated. RST incorporates molecular distances
between alleles, and if RST is substantially higher than h,
then this might indicate that mutations have contributed to
differentiation, indicating a substantial phylogeographical
signal (Slatkin 1995). In the present case, however, h [ RST
Fig. 3 Neighbour-joining dendrogram relating 10 Lupinus alopecuroides populations based on dmu2 distances (Goldstein et al. 1995).
Bootstrap values across loci were based on 10,000 permutations by
locus
suggesting that drift is the predominant force underlying
differentiation. Our results also show that populations of L.
alopecuroides are not in Hardy–Weinberg equilibrium. We
suggest that this disequilibrium is due to selfing and
enhanced genetic drift rather than null alleles, as it seems
unlikely that null alleles should occur at all loci and populations, whereas selfing would be expected to affect all loci.
Finally, we suggest that low genetic diversity within populations and high genetic differentiation between populations
are related to founder effects, lack of gene flow, and/or
autogamy.
Founder effects—in tropical sky islands, populations
are likely founded by individuals arriving from long
distances, rather than from the warm tropical surroundings (Carson and Templeton 1984). Long-distance
dispersal of L. alopecuroides seeds might be limited
since rodents and human activity, important dispersers of
Lupinus elsewhere (Milla and Iriondo 2011; Fagan et al.
2005, Maron and Kauffman 2006; Australian Department of Health and Ageing 2013), are nearly absent in
the super-páramo (Sklenář and Ramsay 2001, Rangel
2006). Therefore, L. alopecuroides populations are likely
founded by a small number of individuals. This produces
severe bottlenecks that cause loss of genetic variation
and increases genetic differentiation (Mayr 1954). Furthermore, Pleistocene climatic oscillation and intensive
volcanic activity could also promote founder and bottleneck events by causing population expansions and
contractions, and by affecting the populations’ extinction–recolonization rate (Carson and Templeton 1984;
Carson et al. 1990; Beheregaray et al. 2003). Repeated
founder events may have triggered genetic divergence
among populations (Clegg et al. 2002), and loss of
genetic variation through sequential bottlenecks, as has
previously been invoked to explain low variation in
other species (e.g. Hedrick 1996). Interestingly, COCUY
is the only population that is not located on a volcanic
mountain massif (Kroonenberg et al. 1990) and it
showed the highest genetic diversity. However, further
research is needed to test the influence of volcanic
activity and Pleistocene climatic oscillations on withinpopulation genetic variation.
Lack of gene flow—in isolated populations, genetic drift
leads to fixation of random alleles, and hence to loss of
genetic variation and high genetic differentiation due to the
absence of gene flow (Lesica and Allendorf 1995; Frankham
et al. 2002; Allendorf and Luikart 2006; Bech et al. 2009).
The high h value in our data indicates that populations of L.
alopecuroides are fixed for different alleles, suggesting an
absence of gene flow.
Autogamy—low within-population diversity and high
differentiation among populations are often observed in
selfing species (Hamrick and Godt 1996). In L.
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alopecuroides, high rates of selfing may be expected due to
low availability of pollinators (Vásquez personal observation). Autogamous self-fertilization, which is common
among islands plants (Carlquist 1974; Baker 1967), provides
reproductive assurance when outcross pollination is limited
by low availability of mates and/or pollinators (Karoly
1992). In the Venezuelan highlands, Berry and Calvo (1989)
found near absence of pollinators and higher levels of selfing
in four species of Espeletia (Asteraceae) growing above
4000 m a.s.l. Like lupines, Espeletia species are mainly
pollinated by bumblebees. Significant decrease of diversity
and activity levels of pollinators with elevation was also
found in the Chilean Andes (Arroyo et al. 1985). Although,
high inbreeding was indicated in the COCUY population
and self-sterility is rare among lupines (East 1940; Kittelson
and Maron 2000), further research is needed to demonstrate
autogamy in L. alopecuroides and its role in determining the
populations genetic structure.
Genetic relationships between populations
The neighbour-joining phenogram (Fig. 3) identified a
well-supported group consisting of all Ecuadorian populations except ANTISANA. Within that group,
CAYAMBE and SINCHOLAGUA, which were shown to
be genetically indistinguishable (Online Resource 1), are
the only populations located on the eastern Ecuadorian
Cordillera. The population ANTISANA was shown to
differ genetically from the rest of the populations. Interestingly, this population was also phenotypically different
from the rest. In comparison with other populations,
individuals have smaller leaves and inflorescence, and the
overall size of the plants was also smaller (Vásquez,
personal observation).
Finally, correlation between genetic and geographic
distances of pair of populations was not significant, as was
shown by the test for isolation by distance. This indicates
that the genetic structure of L. alopecuroides populations is
not the simple product of geographic spatial structure,
pointing towards the importance of colonization history and
founder effects in driving differentiation among populations. Although it has been heavily debated, the role of
founder effects in evolution remains poorly understood
(Clegg et al. 2002; Spurgin et al. 2014). It is unclear if
founder effects can persist through evolutionary time when
at the same time ongoing selection, mutation, gene flow and
drift affect the genetic composition of populations (Hoeck
et al. 2010; Spurgin et al. 2014). In this regard, it has been
suggested that, depending on the severity and the continuality of the founder effects (Clegg et al. 2002), and on the
extent of gene flow and selection, founder effects may be
detectable in present populations, and may play an important
role in the initial stages of speciation (Spurgin et al. 2014).
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Possible effects of microsatellite cross-species
amplification
Success of cross-species transfer of microsatellite markers
depends on the evolutionary distance between the source
and the target species (Rossetto 2001; Selkoe and Toonen
2006; Barbara et al. 2007). Lupinus alopecuroides, L.
microcarpus, and L. nanus belong to a large western New
world ‘‘super-radiation’’ (5.0–13.2 Ma) that comprises the
western lupines from North America, Mexico and the Andes
(Drummond et al. 2012). Low sequence divergence in
adaptive radiations allows transfer of polymorphic
microsatellite markers between species of the same subfamily and beyond (Barbara et al. 2007; Bezault et al. 2012).
Otherwise, in plants, cross-species transfer of polymorphic
markers is likely to be successful mainly within genera
(Rossetto 2001; Barbara et al. 2007).
Cross-species transfer of microsatellites markers is often
accompanied by a decrease in allelic diversity (Selkoe and
Toonen 2006). However, if cross-species amplification
underlies the low variation observed in the present study,
then this should be a species-wide effect, whereas low
variation within populations but higher variation in the
species as a whole would suggest that low variation reflects
demographic history. The latter was the case in our study;
the total number of alleles for the species ranged from 2 to 6
across loci (Online Resource 1). One population (COCUY)
in fact exhibited most of the observed alleles, whereas the
other populations were mostly fixed for different alleles.
We, therefore, conclude that the observed patterns of
microsatellite DNA variation reflect genuine demographic
processes and are not a result of reduced variation due to
cross-species transfer.
Conclusions
Our study reveals extremely low genetic diversity within
populations and high genetic differentiation between populations of L. alopecuroides across its range. We suggest that
this pattern is related to founder effects, lack of gene flow,
and possibly autogamy. However, further research is needed
to provide evidence for autogamy in L. alopecuroides. The
genetic relationships between the populations and the lack of
correlation between genetic and geographic distances point
to the importance of colonization history and founder effects
in determining the populations’ genetic structure. Although
based on a limited number of markers, our study gives
insights into the evolution of the unexplored but fascinating
sky island Andean flora. Moreover, L. alopecuroides provides an exceptional opportunity for understanding the role
of founder effects in evolution. Future studies should focus
on reconstructing the colonization history of L.
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Alp Botany
alopecuroides, including the impact of volcanism and
Pleistocene climatic oscillations on their population dynamics. Moreover, the low genetic variation and supposedly low
adaptive potential in most populations suggest that the species could be particularly susceptible to anthropogenic
disturbance. Formulation of a conservation strategy to protect
L. alopecuroides is, therefore, strongly recommended.
Acknowledgments Thanks to Eva Dušková and Lenka Flašková for
laboratory assistance, and to Jan Brotánek and Vojtěch Zeisek for
help with fieldwork. We also thank Filip Kolář for helpful discussions
and the Fond Mobility of Charles University in Prague for financial
support.
Compliance with ethical standards
Conflict of interest
of interest.
The authors declare that they have no conflict
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